I’m having difficulty achieving high RNA integrity in my samples. Although the 260/280 and 260/230 ratios are satisfactory after RNA extraction, the RNA samples show signs of degradation when analyzed using gel electrophoresis.
Typically, degradation happens if the tissue is allowed to thaw before it's placed in the appropriate buffer OR if you do not take care to keep your samples on ice at all times after isolation.
Seeing a little bit of "smearing" on a gel is normal since RNAs that are being processed have variable lengths. As long as you see strong bands for the rRNAs, you'll be just fine.
I see eye to eye with Katie A S Burnette , the main source of degradation comes from the sample being thawn without buffer, but if you work with plants your starting material may be fresh most of the times.
Then, there are the typical pieces of advice when extracting RNA:
- Clean surfaces and pippetes with RNAse away or similar products
- Wear gloves
- Use filter tips
- Use RNAse free material (reagents, tubes and tips)
- If you have long hair, put it in a ponytail
- Add 1% (v/v) bleach to the agarose gel to ensure your samples are not degraded during the electrophoresis.
Hope some of these help!
Thank you Daniel Martín and Katie A S Burnette for replying. I have done sampling with fresh seedlings for both maize and rapeseed. immediately after weighting I put them on liquid nitrogen and then homogenized the sample and I am using RNeasy Plant Mini Kit to extract the RNA. But I am seeing the degradation. I don't know how I can control that in which step it is happening. Could you give me some hints about it, please?
Do you have an agarose gel image to share?
I assume you are doing the grinding in a liquid-nitrogen cooled mortar?
How are you transferring the ground tissue to the first tube for your RNA extraction? I found that I had the best luck by adding a very small portion of liquid nitrogen to the tissue, swirling to make sure it's all in the liquid, and then very quickly pouring into a liquid nitrogen chilled 1.7ml tube. Then, carefully & quickly hold the tube by the open cap & submerge most of the tube in a bucket of liquid nitrogen until the liquid nitrogen inside the tube has evaporated away. Then you close the lid (if you close it too soon, the expanding N2 gas can make it explode open).
Once all samples are ground & in the liquid nitrogen, then you can add the first buffer from the kit. Make sure the ground tissues DOES NOT thaw at any point before adding the buffer.
Also, check the date that the Beta-mercaptoethanol was added to the buffer, it has a relatively short shelf-life.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA